We undertook this study to evaluate efficacy of the Siemens Novagnost and Enzygnost EBV assays in routine clinical use as an aid to determining EBV exposure and serostatus. The samples were drawn as part of normal clinical practice from patients presenting with symptoms suspicious for EBV infection, or who had been exposed to a person with a known EBV active infection. In our estimation this study group was reasonably representative of populations in industrialized nations at normal risk for EBV infection
, as 89.6% of our patient results demonstrated previous exposure, 7.5% of the patients were determined to be EBV naive, and only 2.9% were ultimately diagnosed with an acute EBV infection.
For EBV serology to be a practical aid in the diagnosis of IM (its most common clinical usage), it should either rule in or rule out an acute infection with a high degree of accuracy following a single blood draw. Additionally, it should also accurately distinguish primary acute infection from past infection, as well as identify those individuals who are EBV naive, and who therefore remain at risk of contracting an EBV infection. Ideally, results should not be confounded by unusual serological profiles that require additional testing to achieve resolution (e.g., isolated VCA IgG, i.e., VCA IgM–/VCA IgG+/EBNA IgG–; isolated EBNA, i.e., VCA IgM–/VCA IgG–/EBNA IgG+; or all markers positive, i.e., VCA IgM+/VCA IgG+/EBNA IgG+). Assays such as Enzygnost and Merifluor, which report only qualitative results for total viral IgM and IgG, are less subject to such confusion. Results from these two assays demonstrated the greatest concordance with EBV-naive status, and thus would be expected to perform reliably for ruling out EBV infection. The Novagnost assay also demonstrated good agreement with EBV-negative status, although concordance was not as good as that achieved by either the Enzygnost or Merifluor assays. This reduced concordance is directly attributable to the larger number of samples that were either incorrectly assigned or indeterminate (4 for Novagnost, versus 0 for Enzygnost and 3 for Merifluor). It is possible that we would have been able to resolve the 4 indeterminate samples had we retested these individuals 2 to 4 weeks later, following the manufacturer’s guidelines; this resolution might possibly have improved the concordance for the Novagnost assay. However, since one of the stated purposes of our study was to determine serostatus on the basis of a single blood draw, only, we elected to not conduct later testing. It is important to note, however, that the manufacturer clearly states that gray zones exist for the Novagnost assays (between 8.0 and 11.5 U) and the Enzygnost IgG assay (between 0.1 and 0.2 U), and recommends retesting patients with sera yielding gray zone values two to four weeks after the original blood draw. If samples continue to report in the gray zone, they should be considered negative. Clinical laboratories should keep this in mind and consider retesting patients accordingly if alternative methods are not available for resolving indeterminate samples.
All three of the assays demonstrated excellent and identical concordance (100%) with the final EBV status for sera from patients with an acute primary infection: all three correctly identified those sera that were either IgM+ only, or were IgM+ in the presence of either total IgG or specific VCA IgG. Although it is not possible to tell if the Enzygnost and Merifluor assays are detecting EBNA-1 IgG as part of the IgG population, the absence of EBNA-1 IgG detection by the Novagnost assay for all 15 acute sera confirms that none of the assays are detecting EBNA-1 in these samples. This suggests that both of the Siemens assays can be considered reliable for detecting a primary acute EBV infection on the basis of a single blood draw. Later retesting to establish a positive diagnosis of infectious mononucleosis is not likely to be necessary unless phlebotomy has occurred so early in the disease process that the patient has not yet developed a sufficient antibody response.
The greatest challenge in EBV testing can lie in differentiating between an acute infection and a past infection, with or without reactivation. The Enzygnost assay performed at least as well as the Merifluor assay in this respect. The Enzygnost assay has also been shown to perform well in other studies
[13, 14], although Bruu et al. noted that because separate EBNA-1 detection is lacking, it is not possible to determine if IgM positivity is a true indication of primary acute infection, or if it might represent reactivation in those cases where EBNA-1 has been lost or was never generated
[8, 15]. Since reactivation is typically thought of as a rare event, however, this is likely of little consequence for diagnosis in the general population.
Atypical serotypes may be observed when using assays such as Novagnost, as they detect EBNA-1 IgG specifically, in addition to IgG to VCA; thus, their interpretation can constitute a diagnostic challenge
[9, 16]. Garcia et al. determined that the isolated EBNA-1 serotype occurred in up to 50% of their patients ultimately diagnosed with a latent EBV infection, and further noted that knowledge of latent infection is especially critical preoperatively in prospective solid organ transplant patients
. In a study by Nystad et al., the simultaneous detection of VCA IgM, VCA IgG and EBNA-1 was associated with primary infection in 42% of their patients, although in at least 23% of the study group it was associated with clinically inconsequential EBV reactivation
. In contrast, Klutts et al. found that 77.8% of patients who were VCA IgM+/VCA IgG+/EBNA-1+ proved to have had a past infection, were recovering from a primary infection, or were manifesting EBV reactivation
. Debyser et al. also associated the triple-positive serotype with reactivation, noting that reactivation can also be indicated by the VCA IgM+/VCA IgG–/EBNA-1+ serotype, but proposed that reactivation is only relevant in patients suffering from (or as in the case with Garcia et al.,
 subject to) immune suppression. Additionally, they suggested that isolated VCA IgG, especially when the IgG titer is high, was indicative of persistent or chronic EBV infection
; however, 69.6% of patients with the VCA IgG+ only serotype in the study by Klutts et al.
 were diagnosed with a past infection.
In our opinion, the isolated VCA IgG+ results generated by the Novagnost assay are the most problematic when screening otherwise presumed healthy individuals. Although, as Klutts suggests, the majority of these likely represent remote infections, it is not possible to tell from the assay results alone if some of these sera might be indicative of a late active infection with early IgM decline and weak EBNA response, or a remote infection in which EBNA IgG has been lost or never generated. Since there is the possibility that individuals with isolated VCA IgG+ could have an acute infection, sera such as these should be tested for VCA IgG avidity or their status should be confirmed by some other method, such as Western blot or PCR
[20–22]. Since the goal of our study was to evaluate only the performance of the microtiter assays as they compared to each other, we did not conduct any further evaluation of any of the indeterminate sera. While this might be considered a limitation of the study, we felt that further testing was not warranted as the original testing indicated 100% concordance between the three methods for detecting acute infection, and in all likelihood, the indeterminate sera truly represented remote infections.
Despite the difficulties that EBNA IgG detection can cause in interpreting rare serotypes, it is still considered to be a valuable component of EBV testing; absence of EBNA IgG in the presence of VCA IgM is considered definitive for ruling in acute infection, while the presence of EBNA IgG typically rules out acute infection, even if VCA IgM antibodies persist. Although, as shown by Nystad and Klutts in the above discussion, EBNA-1 can be indicative of a resolving infection. VCA IgG positivity, however, can be equally attributable to acute, resolving, or remote infections. Furthermore, VCA IgG levels may fall to undetectable levels in some individuals following acute infection, while EBNA IgG generally persists for life
In our hands, the Novagnost assay performed about as well as both the Enzygnost and Merifluor assays for detecting active primary infection, but because the Novagnost assay reports the presence of antibodies to EBNA-1 in addition to the VCA antibodies, it was more difficult to appropriately classify all past infections. Of the total 464 sera determined to represent past infections, 65 expressed atypical serotypes and were subsequently classified as indeterminate. However, the 55 samples demonstrating isolated VCA IgG+ status constitute approximately 12% of the study population, which is consistent with the expected percentage of individuals who either never develop EBNA-1 antibodies or in whom antibody production is lost. Since VCA IgM rarely persists beyond acute infection, it is likely that these 55 samples do, indeed, represent patients with remote exposure to EBV, although it cannot be determined if these sera represent reactivation or chronic infection. Additionally, eight samples were positive only for EBNA-1 IgG, while two samples were positive for all markers; either of these profiles could be interpreted as past infection as shown in Table
2, and by the arguments presented by other researchers cited in the previous paragraphs.